There is a continuing need in medical practice, research and diagnostic procedures for rapid and accurate detection or quantification of biological and chemical substances which are present in biological fluids, cells or tissues. For example, the presence of drugs, hormones, steroids, polypeptides, nucleotides, prostaglandins, proteins, carbohydrates or infectious organisms (bacteria, fungi or viruses) in biological specimens has to b determined in an accurate and rapid fashion for suitable diagnosis or treatment.
For example, organisms classified as gram positive bacteria, such as group specific Streptococcus, are known to be pathogenic in humans. Group A organisms are primarily responsible for causing B hemolytic pneumonia, scarlet fever, rheumatic fever, cardiac sequelae, glomerulo nephritis, septic sore throat and puerpueral sepsis. Because of the serious nature of infections potentially caused by Streptococcus A, it is important to diagnose its presence at an early stage so that an appropriate course of treatment can be pursued. In other words, it is highly desirable to have a highly sensitive assay so that infectious agents can be detected at low concentration.
To provide diagnostic determinations, various methods have been devised for isolating and identifying biological or chemical substances employing specific binding reactions between the substance to be detected (identified as a "target ligand" or simply "ligand" herein) and receptors (molecules which specifically react or bind with that substance). This reaction between a ligand and its corresponding receptor is known as a specific binding reaction. Where either the ligand or receptor is an antibody, the reaction is known as an immunological reaction. More than one ligand or receptor may participate in each reaction.
Such reactions are detected in a number of ways. Generally, one or more participants in the specific binding reaction is detectably labeled. That is, it is either chosen because it is inherently detectable, or a detectable moiety (for example, an enzyme, radioisotope, chromogen or fluorogen) is incorporated therein in some manner. Many assays (for example, ELISA) today utilize enzymes as detectable moieties because of the convenience they provide in requiring minimum equipment and skills needed for the assays, as well as improved sensitivity is some cases.
One significant problem encountered in many analytical procedures which utilize specific binding reactions is the occurrence of non-specific binding reactions. For example, specific binding species, such as antibodies or antigens, may indiscriminately react with other proteins, carbohydrates or chemical or biological materials for which they are not specific. They may also react with each other and clump together thereby inhibiting specific binding with a molecule for which they have specific reactivity. In addition, if the assays are carried out using solid supports of some type (such as membranes, glass tubes, plates, beads or fibers), the specific binding species may also non-specifically bind to such materials because of the nature of chemical groups on the surfaces.
Polyamide membranes, in particular, are susceptible to non-specific interactions with specific binding species (such as antibodies). It is known that non-specific binding to solid surfaces (such as polyamides) can be minimized by coating them with proteins, such as casein or bovine serum albumin.
All of these undesired non-specific reactions cause background interference (that is, unwanted detectable signal) and poor assay sensitivity (that is, detection of low concentrations is poor). In copending and commonly assigned U.S.S.N. 098,433 (filed Sept. 18, 1987, now U.S. Pat. No. 4,828,980, by Snyder, Warren III and Nelson), it is disclosed that non-specific interactions in agglutination assays can be minimized by coating microporous membranes used in those assays with certain low pI proteins or carbohydrates. While an improvement was achieved with agglutination assays, it has been found that coating membranes similarly in non-agglutination assays, such as ELISA or sandwich assays, does not acceptably provide both low background and high assay sensitivity. Sensitivity in assays using enzyme labels is generally high, but unwanted background can be a problem even if the membrane is coated.
It is highly desirable to have assays which are highly sensitive to low concentrations of ligand but which also exhibit minimal unwanted signal from non-specific interactions.